20X SSC, pH 5.3
Mix thoroughly 66 g 20X SSC in 200 mL purified H2O. Adjust to pH 5.3 at ambient temperature using concentrated HCl and QS to final volume of 250 mL. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.
Add 49 mL formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O to a glass Coplin jar and mix thoroughly. Verify that the pH is 7.0 -7.5 by measuring the pH at ambient temperature. Between use, store covered at 4ºC. Discard after 7 days.
Ethanol wash solutions (70%, 85% and 100%)
Dilute 100% ethanol v/v with purified H2O to prepare the wash solutions. Between uses, store covered at ambient temperature. Discard stock solutions after 6 months.
0.4X SSC/0.3% NP-40 Wash Solution
Mix thoroughly 20 mL of 20X SSC with 950 mL purified H2O. Add 3 mL NP-40. Mix thoroughly until NP-40 is dissolved. Adjust pH to 7.0 - 7.5 with NaOH. Add purified H2O to bring final volume to 1 L. Store at ambient temperature. Discard stock solution after 6 months, or if solution appears cloudy or contaminated.
2X SSC/0.1% NP-40 Wash Solution
Add 1 mL NP-40. Adjust pH to 7.0 - 7.5 with NaOH. Add purified H2O to bring final volume to 1 L. Store at ambient temperature. Discard stock soultion after 6 months, or if solution appears cloudy or contaminated.
Direct-label the test (and unlabeled control) DNA with SpectrumGreenTM dUTP. See the Vysis CGH Nick Translation Kit for the procedure.
Positive and negative controls provide comparisons for evaluating the hybridization and interpretation of the CGH data. For a negative control, use normal DNA for both the test and reference DNA (Cat. No.32-804024, 32-802024). The intensity profiles for this experiment should be within the threshold values as determined by image analysis. For a positive control, use test DNA (Cat. No. 32-800227) that is extracted from a cell line with known genetic aberrations that are easy to detect by CGH analysis.
Normal Metaphase CGH Target Slides
Do not pretreat slides. Slides are prepared using standard cytogenetic slide preparation methods that are optimized for CGH. The slides are made from phytohemagglutinin(PHA) stimulated lymphocytes cultured for 48 to 72 hours before thymidine is added to synchronize the cells. The chromosome length is 400 - 550 bands. The lymphocytes are derived from a karyotypically normal male donor.
Preparing the Probe Mix
To produce a hybridization with signal intensities equivalent for both SpectrumGreenTM and SpectrumRedTM labeled DNA, the amount of SpectrumGreen labeled DNA is increased.
Hybridizing the Probe to the Target Metaphase
Washing the Slide
Visualizing the Hybridization
Apply 10 µL of DAPI II counterstain and a coverslip to each hybridization location.
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